Consensus recommendations for using the Multiplate(®) for platelet function monitoring before cardiac surgery.

Patients requiring urgent cardiac surgery are usually already taking antiplatelet drugs including aspirin and a P2Y12 ADP receptor antagonist (e.g., clopidogrel, prasugrel or ticagrelor). This presents clinicians with the challenge of balancing the risk of thrombotic complications, if antiplatelet drugs are stopped before surgery, with the problems of excessive bleeding when surgery is performed in the absence of adequate platelet function. Preoperative platelet function monitoring is able to identify when patients have recovered platelet function. The Multiplate(®) (multiple electrode impedance platelet aggregometer) is a point of care device that enables monitoring of platelet function. The authors offer recommendations based on real-world, collective experience in the use of platelet function monitoring. These cover the use of the Multiplate(®) analyser to predict the need for platelet transfusion in the perioperative period and the individualized waiting period after cessation of P2Y12 ADP receptor antagonists before cardiac surgery.

The creation and validation of the Jamaica Personality Disorder Inventory.

OBJECTIVE: To describe the creation and validation of the Jamaica Personality Disorder Inventory (JPDI) screening questionnaire. METHOD: Using the phenomenological triad of power management, dependency and psychosexual issues, drafts of the JPDI were piloted on patients from psychiatric and medical wards. The JPDI consisted of 38 close-ended, yes/no questions. Validation was conducted in a sample of 200 patients, using the International Personality Disorder Examination-Screening Instrument (IPDE-S), the Brief Screen for Depression and consultant psychiatrists' Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) personality disorder interview. Construct validity was assessed through principal component factor analysis; Spearman correlation was used to assess criterion-related and discriminant validity; Cronbach's alpha was used to assess reliability of the entire scale as well as the resulting factors. The Multitrait Multimethod Matrix (MTMM) was used to assess discriminant and construct validity. RESULTS: Factor analysis revealed eight clusters consisting of 30 of the 38 questions, which had close congruence with the clinical triad. Cronbach's alpha for the entire scale was α = 0.79, ranging from a high 0.70 to 0.82 to low 0.63 to 0.45. The JPDI exhibited a sensitivity of 95.06% and a specificity of 67.71%. Significant correlation of scores for the JPDI and IPDE-S (r = 0.432, p = 0.000) and the JPDI and the DSM IV-TR diagnosis (r = 0.598, p = 0.000) established concurrent validity for the JPDI. Correlations (r = 0.293, p = 0.000) suggested that the JPDI possessed predictive validity. The complete sample matrix of the MTMM provided evidence of both convergent and discriminant validity, and thereby, construct validity. CONCLUSION: The JPDI demonstrated reliability, and criterion-related and discriminant validity.

The creation and validation of the Jamaica personality disorder inventory / Creación y validación del inventario de trastornos de la personalidad en Jamaica

OBJECTIVE: To describe the creation and validation of the Jamaica Personality Disorder Inventory (JPDI) screening questionnaire. METHOD: Using the phenomenological triad of power management, dependency and psychosexual issues, drafts of the JPDI were piloted on patients from psychiatric and medical wards. The JPDI consisted of 38 close-ended, yes/no questions. Validation was conducted in a sample of 200 patients, using the International Personality Disorder Examination-Screening Instrument (IPDE-S), the Brief Screen for Depression and consultant psychiatrists' Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) personality disorder interview. Construct validity was assessed through principal component factor analysis; Spearman correlation was used to assess criterionrelated and discriminant validity; Cronbach's alpha was used to assess reliability of the entire scale as well as the resulting factors. The Multitrait Multimethod Matrix (MTMM) was used to assess discriminant and construct validity. RESULTS: Factor analysis revealed eight clusters consisting of 30 of the 38 questions, which had close congruence with the clinical triad. Cronbach's alpha for the entire scale was α = 0.79, ranging from a high 0.70 to 0.82 to low 0.63 to 0.45. The JPDI exhibited a sensitivity of 95.06% and a specificity of 67.71%. Significant correlation of scores for the JPDI and IPDE-S (r = 0.432, p = 0.000) and the JPDI and the DSM IV-TR diagnosis (r = 0.598, p = 0.000) established concurrent validity for the JPDI. Correlations (r = 0.293, p = 0.000) suggested that the JPDI possessed predictive validity. The complete sample matrix of the MTMM provided evidence of both convergent and discriminant validity, and thereby, construct validity. CONCLUSION: The JPDI demonstrated reliability, and criterion-related and discriminant validity.

OBJETIVO: Describir la creación y validación del cuestionario de tamizaje del Inventario de Trastornos de la Personalidad en Jamaica (JPDI). MÉTODO: Usando la tríada fenomenológica de manejo del poder, dependencia y problemas psicosexuales, se realizaron pruebas pilotos usando versiones p rovisionales del JPDI con pacientes de salas médicas y psiquiátricas. El JPDI constaba de 38 preguntas cerradas, del tipo que requieren sí o no. La validación se realizó con una muestra de 200 pacientes, usando el Instrumento de Tamizaje del Examen Internacional de los Trastornos de Personalidad (IPDE-S), la Prueba Breve para la Depresión, y el Manual Diagnóstico y Estadístico de los Trastornos Mentales, cuarta edición (DSM-IV) de los psiquiatras consultantes, para entrevistas de trastornos de personalidad. La validez de constructo se evaluó a través de análisis factorial de componentes principales. El coeficiente de correlación de Spearman se utilizó para evaluar la validez de criterio y la validez discriminante. El coeficiente Alfa de Cronbach fue utilizado para evaluar la fiabilidad de toda la escala, así como los factores resultantes. La matriz multirasgo-multimétodo (MTMM) fue utilizada para evaluar la validez de constructo y la validez discriminante. RESULTADOS: El análisis factorial reveló ocho clústeres que constaban de 30 de las 38 preguntas, las cuales presentaban una estrecha congruencia con la tríada clínica. El Alfa de Cronbach para toda la escala fue α = 0.79, fluctuando desde valores altos de 0.70 a 0.82 hasta valores bajos de 0.63 a 0.45. El inventario JPDI mostró una sensibilidad de 95.06% y una especificidad de 67.71%. La correlación significativa de las puntuaciones para el JPDI y el IPDE-S (r = 0.432, p = 0.000) y el JPDI y el diagnóstico de DSM IV-TR (r = 0.598, p = 0.000) estableció una validez concurrente para el JPDI. Las correlaciones (r = 0.293, p = 0.000) sugirieron que el JPDI poseía validez predictiva. La matriz completa de la muestra de la MTMM proporcionó evidencia tanto de la validez discriminante como de la validez convergente, y por ende, de la validez de constructo. CONCLUSIÓN: El inventario JPDI demostró fiabilidad, así como validez de criterio y validez discriminante.

Permanent pacemaker insertion after CoreValve transcatheter aortic valve implantation: incidence and contributing factors (the UK CoreValve Collaborative).

BACKGROUND: Permanent pacemaker (PPM) requirement is a recognized complication of transcatheter aortic valve implantation. We assessed the UK incidence of permanent pacing within 30 days of CoreValve implantation and formulated an anatomic and electrophysiological model. METHODS AND RESULTS: Data from 270 patients at 10 centers in the United Kingdom were examined. Twenty-five patients (8%) had preexisting PPMs; 2 patients had incomplete data. The remaining 243 were 81.3±6.7 years of age; 50.6% were male. QRS duration increased from 105±23 to 135±29 milliseconds (P<0.01). Left bundle-branch block incidence was 13% at baseline and 61% after the procedure (P<0.001). Eighty-one patients (33.3%) required a PPM within 30 days. Rates of pacing according to preexisting ECG abnormalities were as follows: right bundle-branch block, 65.2%; left bundle-branch block, 43.75%; normal QRS, 27.6%. Among patients who required PPM implantation, the median time to insertion was 4.0 days (interquartile range, 2.0 to 7.75 days). Multivariable analysis revealed that periprocedural atrioventricular block (odds ratio, 6.29; 95% confidence interval, 3.55 to 11.15), balloon predilatation (odds ratio, 2.68; 95% confidence interval, 2.00 to 3.47), use of the larger (29 mm) CoreValve prosthesis (odds ratio, 2.50; 95% confidence interval, 1.22 to 5.11), interventricular septum diameter (odds ratio, 1.18; 95% confidence interval, 1.10 to 3.06), and prolonged QRS duration (odds ratio, 3.45; 95% confidence interval, 1.61 to 7.40) were independently associated with the need for PPM. CONCLUSION: One third of patients undergoing a CoreValve transcatheter aortic valve implantation procedure require a PPM within 30 days. Periprocedural atrioventricular block, balloon predilatation, use of the larger CoreValve prosthesis, increased interventricular septum diameter and prolonged QRS duration were associated with the need for PPM.

Use of respiratory function tests to predict survival in amyotrophic lateral sclerosis.

Respiratory function tests (RFTs) are commonly used as a measure of progression in ALS. This study assessed the ability of various RFTs to predict survival in ALS patients. Subjects with ALS had one or more measurements of seated and supine FVC, maximal inspiratory pressure (MIP) and maximal expiratory pressure (MEP). Kaplan-Meier (KM) analysis was used to determine whether patients with abnormal RFTs had shorter survival than those with normal RFTs. The sensitivity and specificity of RFTs as predictors of two-year survival were calculated from receiver operating characteristic (ROC) curves. With KM analysis, subjects with abnormal values of seated FVC, supine FVC, MIP and MEP had significantly reduced survival compared to subjects with normal values. With ROC curves, a normal supine FVC was highly predictive for two-year survival and had superior sensitivity over seated FVC. Slower rates of decline in seated or supine FVC were strong predictors of two-year survival. Our study demonstrates that respiratory function measurements are useful to predict survival in ALS patients. We show that measurements of FVC in the supine position are worth including in the assessment of respiratory function in ALS.

Ultra scale-down approach to correct dispersive and retentive effects in small-scale columns when predicting larger scale elution profiles.

Ultra scale-down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale-down mimics must predict large-scale process performance accurately. For example, one application of a scale-down model involves using it to predict large-scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large-scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale-down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale-down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory- and 18.3 L pilot-scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger-scale elution profiles. These data then provide the justification to adjust small-scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large-scale and which can then be used for subsequent ultra scale-down operations.

Obesity pharmacotherapy from a regulatory perspective: overview and key challenges.

Obesity is an epidemic with tremendous impact on both patients and health-care systems globally. This paper explores some of the questions related to the clinical development of new pharmacotherapies in the context of an evolving regulatory perspective. These include patient entry criteria, clinical database size, study designs, weight loss end points (including those for maintenance of weight loss and prevention of weight regain), clinically important patient-reported outcomes, comorbidity/risk factor end points, and challenges in establishing safety and efficacy in adolescent/pediatric patients, and approaches to the development of combination pharmacotherapies. Ultimately, patients, physicians, academia, industry, payers, and governments must continue to partner with regulators to help establish the appropriate balance between the known adverse consequences associated with inadequate treatment of the growing obesity epidemic and the concern for potential unknown risks that may be associated with the long-term use of new pharmacotherapies.

Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification.

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.

Susceptibility of stromelysin 1-deficient mice to collagen-induced arthritis and cartilage destruction.

OBJECTIVE: It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage. METHODS: The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341. RESULTS: SLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice. CONCLUSION: Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.

Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase activity in normal, osteoarthritic, and rheumatoid joints.

To examine the activity of matrix metalloproteinases (MMPs) and aggrecanase in control and diseased human articular cartilage, metabolic fragments of aggrecan were detected with monospecific antipeptide antibodies. The distribution and quantity of MMP-generated aggrecan G1 fragments terminating in VDIPEN341 were compared with the distribution of aggrecanase-generated G1 fragments terminating in NITEGE373. Both types of G1 fragments were isolated from osteoarthritic cartilage. The sizes were consistent with a single enzymatic cleavage in the interglobular domain region, with no further proteolytic processing of these fragments. Both neoepitopes were also detected by immunohistochemistry in articular cartilage from patients undergoing joint replacement for osteoarthritis (OA), rheumatoid arthritis (RA), and in cartilage from adults with no known joint disease. In control specimens, the staining intensity for both G1 fragments increased with age, with little staining in cartilage from 22-wk-old fetal samples. There was also an increase with age in the extracted amount of MMP-generated neoepitope in relation to both aggrecan and collagen content, confirming the immunohistochemical results. After the age of 20-30 yr this relationship remained at a steady state. The staining for the MMP-generated epitope was most marked in control cartilage exhibiting histological signs of damage, whereas intense staining for the aggrecanase-generated fragment was often noted in adult cartilage lacking overt histological damage. Intense staining for both neoepitopes appeared in the more severely fibrillated, superficial region of the tissue. Intense immunostaining for both VDIPEN- and NITEGE- neoepitopes was also detected in joint cartilage from patients with OA or RA. Cartilage in these specimens was significantly more degraded and high levels of staining for both epitopes was always seen in areas with extensive cartilage damage. The levels of extracted VDIPEN neoepitope relative to collagen or aggrecan in both OA and RA samples were similar to those seen in age-matched control specimens. Immunostaining for both types of aggrecan fragments was seen surrounding the cells but also further removed in the interterritorial matrix. In some regions of the tissue, both neoepitopes were found while in others only one was detected. Thus, generation and/or turnover of these specific catabolic aggrecan fragments is not necessarily coordinated. Our results are consistent with the presence in both normal and arthritic joint cartilage of proteolytic activity against aggrecan based on both classical MMPs and "aggrecanase."

Expression and immunoaffinity purification of human inducible nitric-oxide synthase. Inhibition studies with 2-amino-5,6-dihydro-4H-1,3-thiazine.

Recombinant human inducible nitric-oxide synthase (rH-iNOS) was expressed in the baculovirus system and purified by a novel immunoaffinity column. rH-iNOS and its native counterpart from cytokine-stimulated primary hepatocytes exhibited similar molecular mass of 130 kDa on SDS-polyacrylamide gel electrophoresis, recognition by antipeptide antibodies, specific activities, and IC50 values for inhibitors. The active dimeric form exhibited a specific activity range of 114-260 nmol/min/mg at 37 degrees C and contained 1.15 +/- 0.04 mol of calmodulin/monomer. The enzyme exhibited a Soret lambdamax at 396 nm with a shoulder at 460 nm and contained 0. 28-0.64 mol of heme/monomer. Dithionite reduction under CO yielded an absorbance maximum at 446 nm, indicating a P450-type heme. Imidazole induced a type II difference spectrum, reversible by L-Arg. 2-Amino-5,6-dihydro-4H-1,3-thiazine (ADT) was competitive versus L-Arg (Ki = 22.6 +/- 1.9 nM), reversed the type II difference spectrum induced by imidazole (Kd = 17.7 nM), and altered the CO-ferrous absorbance of rH-iNOS. L-Arg did not perturb the CO-ferrous adduct directly, but it partially reversed the ADT-induced absorbance shift, indicating that both bind similarly to the protein but interact differently with the heme.

Inducible nitric oxide synthase requires both the canonical calmodulin-binding domain and additional sequences in order to bind calmodulin and produce nitric oxide in the absence of free Ca2+.

All three mammalian isoforms of nitric oxide synthase (NOS) must bind calmodulin (CaM) for enzymatic activity. Only NOS2 (the inducible isoform, iNOS) does so at the low levels of free Ca2+ in resting cells and when almost all Ca2+ is chelated in cell-free preparations. To test directly whether the predicted CaM-binding region of mouse NOS2 accounts for its Ca2+ independence, we prepared chimeric NOS's in which mouse NOS2 residues 503-532 were reciprocally exchanged with the corresponding residues 725-754 of rat NOS1 (neuronal NOS). Unlike either parent, both chimeras required an intermediate level of free Ca2+ to bind CaM and generate NO. In cell lysates, the concentration of Ca2+ necessary for half-maximal activity (EC50) was approximately 0 for NOS2, 200-300 n for NOS1, and 7-10 n for the chimeras. Results were similar when the region exchanged was enlarged by 7-8 residues toward the amino terminus. In contrast, when the carboxyl-terminal half of NOS2 (residues 454-1144) was replaced with that of NOS1 (residues 675-1429), the resulting chimera resembled NOS1 (EC50, 200-300 n free Ca2+). Truncation analysis suggested that NOS2 residues within the sequence 484-726 were required for Ca2+-independent CaM-binding. Thus, both the canonical CaM-binding domain and additional residues within the region 484-726 are necessary for NOS2's ability to bind CaM and produce NO when Ca2+ levels approach zero.

Active-site structure analysis of recombinant human inducible nitric oxide synthase using imidazole.

Nitric oxide synthase catalyzes the pyridine nucleotide-dependent oxidation of L-arginine to nitric oxide and L-citrulline. It is a specialized cytochrome P450 monooxygenase that is sensitive to inhibition by imidazole. Steady-state kinetic studies on recombinant human inducible nitric oxide synthase (rH-iNOS) demonstrate that imidazole and 1-phenylimidazole are competitive and reversible inhibitors versus L-arginine. Structure-activity relationship and pH dependence studies on the inhibition suggest that the neutral form of imidazole may be the preferred species and that the only modifications allowed without the loss of inhibition are at the N-1 position of imidazole. Optical spectrophotometric studies of rH-iNOS with imidazole and 1-phenylimidazole yielded type II difference spectra exhibiting Kd values of 63 +/- 2 and 28 +/- 3 microM, respectively. These values were in good agreement with the steady-state Ki of 95 +/- 10 and 38 +/- 4 microM, respectively, and confirms the site of binding is at the sixth axial ligand of the heme. Imidazole (2.2 mM) also perturbed the Kd of L-arginine from 3.03 +/- 0.45 to 209 +/- 10 microM. The observed increase in the Kd for L-arginine is consistent with imidazole being a competitive inhibitor versus L-arginine. The IC50 values of imidazole and 1-phenylimidazole were lower in the absence of exogenous BH4, and both inhibitors also competitively inhibited the BH4-dependent activation of the enzyme. These data taken together suggest that the L-arginine, dioxygen, and the BH4 binding sites are in close proximity in rH-iNOS. Furthermore, these studies demonstrate the usefulness of imidazole compounds as active site probes for recombinant human iNOS.